The present invention relates to methods and pharmaceutical compositions for delivering a pharmaceutical agent into a target organ which is protected by a blood barrier and, more particularly, to the delivery of pharmaceutical agents to the brain.
While the invention will be described herein in more detail with respect to delivery of pharmaceutical agents to the brain, it is to be understood that the invention is applicable for delivery of pharmaceutical agents to any organ protected by a barrier.
There are four known mammalian blood barriers including the blood brain barrier (BBB), the blood retinal barrier, the blood testes barrier and the blood mammary gland barrier. All of these function to separate the organ or tissue from features in the periphery, allowing only selective transport of factors. Structurally, these barriers are characterized by tight junctions between the cells and are all endowed with several specific markers such as the glucose transporter, P-glycoprotein, and γ glutamyl transpeptidase. The barriers also contain a paucity of endocytic vesicles within them and the presence of the molecules that regulate the ionic and metabolic gradients that constitute the barrier.
Delivery of many therapeutic agents, particularly nonlipophilic drugs, to these four organs is restricted by their respective barriers.
Over the years, several strategies to circumvent the BBB have been proposed, such as by transient osmotic opening of the BBB, high dosing (e.g., of chemotherapy), use of carrier systems such as antibodies, or even biodegradable implants. However, some of these methods are associated with two major pitfalls: they are invasive procedures and they are relatively inefficient, resulting in only superficial distribution of the drug in the brain. High dosing of pharmaceutical agents to overcome this inefficiency leads to an increase in serious potential side effects. An alternative strategy of delivering drugs across the BBB is by administering their lipophilic precursors (i.e., pro-drugs). However, increased lipophilicity tends to increase uptake by other tissues with resultant toxicity.
Several synthetic NP polymers, arranged as spheres have been studied as carriers of drugs across the BBB. Poly(butyl cyanoacrylate) has been reported to effectively deliver different drugs, including peptides [Kreuter J. Adv. Drug Delivery Rev. 2001, 47:65-81; Gulayev A E, et al., Pharm Res 1999, 16:1564-9]. The formulation efficiency has been attributed to a detergent Tween 80 (polysorbate) coating, which may prevent NP aggregation and delay NP removal by the Mononuclear Phagocyte System (MPS, formerly known as the RES, reticuloendothelial system) [Kreuter J. Adv. Drug Delivery Rev. 2001, 47:65-81; Troster S D et al., Int. J. Pharm. 1990, 61:85-100]. While the exact mechanism of NP brain delivery is unknown, it has been suggested that brain delivery by NPs might be mediated simply by the disruption of the BBB by surfactants i.e., detergents [Lockman P R, et al. Pharm Res 2003; 20:705-13; Olivier J C et al., Pharmaceutical Research 1999; 16:1836-42].
However, recent studies suggest that the effect of polysorbate 80 is unrelated to BBB toxicity, but is rather due to adsorption of blood apolipoproteins, such as apolipoprotein E onto the nanoparticle surface. The particles subsequently appear to mimic low density lipoprotein (LDL) particles and can thereby interact with the LDL receptor leading to their uptake by the endothelial cells via receptor-mediated endocytosis [Kreuter J, et al., Pharm. Res. 2003; 20:409-16; Kreuter J. et al., J. Drug Targeting 2002; 10:317-25]. Other mechanisms such as tight junction modulation or P-glycoprotein inhibition also may occur. Moreover, these mechanisms may run in parallel or may be cooperative thus enabling drug delivery to the brain.
It has also been suggested that liposomes can enhance drug delivery to the brain across the blood-brain barrier [Umezawa and Eto, Biochem Biophys Res. Comm. 153:1038-1044 (1988); Chan et al., Ann. Neurol, 21:540-547 (1987); Laham et al., Life Sciences 40:2011-2016 (1987); and Yagi et al., J. APRlo Biocheme 4:121-125 (1982)]. Liposomes are small vesicles (usually submicron sized) comprised of one or more concentric bilayers of phospholipids separated by aqueous compartments. There is, however, conflicting evidence whether liposomes may be used to enhance the uptake of drugs into the brain. In some cases, it has been shown that liposomes do not cross the blood-brain barrier [Schackert et al., Select. Cancer TheraReut., 5:73-79 (1989) and Micklus et al., Biochem Biophys Acta 1124:7-12 (1992)]. Micklus notes that liposomes circulating in the plasma are ultimately taken up by the liver, digested and the lipid components released and redistributed to other organs [Derksen et al., Biochim. Biophys Acta 971:127-136 (1988)]. The radioactivity found in the brain following intravenous injection of labeled liposomes of Umezawa and Eto, and others may in fact be derived from digested lipids and not from the intact liposomes themselves.
In several cases where liposomes were shown to cross the BBB, the permeability of the BBB itself was disrupted [Rousseau et al., 1997, Magma 5 213-22; Rousseau et al., 1999, Experimental brain research; Experimentelle Hirnforschung; Experimentation cerebrale 125 (1999) 255-64; Stavaraky et al., Magn Reson Imaging 11 (1993) 685-9]. Thus, in these cases the penetration of the liposomes through the BBB cannot be attributed to the liposomes themselves but rather to the disrupted properties of the barrier.
It has been suggested that the use of an external ligand such as mannose can improve a liposomal particle's ability to cross the BBB [Huitinga et al., J exp Med 172 (1990) 1025-33; Umezawa F., Biochem Biophys Res Commun 153 (1988) 1038-44]. A possible penetration mechanism could be that BBB cells and glial cells recognize mannose molecules on the liposome membrane surface. Indeed, the research by Huitinga suggests that only mannosylated liposomes are able to cross the BBB since non-mannosylated liposomes could not penetrate the barrier. It should be noted, that the mannosylated liposomes that were able to cross the barrier remained localized to this area and were not detected in other brain areas [Huitinga et al., 1995, Clin Exp Immunol 100, 344-51]. Also, the mannosylated liposomes were shown to be incorporated in glial cells as opposed to neuronal cells, the former having a receptor for mannose [Umezawa F., Biochem Biophys Res Commun 153, 1988, 1038-44]. PCT Application, Publication No. WO9402178A1 to Micklus discusses the coupling of liposomes to an antibody binding fragment which binds to a receptor molecule present on the vascular endothelial cells of the mammalian blood-brain barrier.
In conclusion, from the presented evidence it remains unclear whether unmodified liposomes are able to cross an undisrupted blood brain barrier, although the use of liposomes modified with external ligands such as mannose and antibody binding fragments do appear to enhance liposome penetration.
Until recently, the brain was considered an immunologically privileged organ [Miller D W, J Neurovirol 1999; 5:570-8]. This was based on early studies that found few antigen-presenting cells in the central nervous system. In addition, there was a perceived lack of a lymphatic system within the brain to carry immunogenic material in the central nervous system to lymph nodes where a humoral immune response could be initiated. The presence of the BBB was thought to prevent the entry of immune cells from the peripheral circulation into the brain. However, there is increasing evidence suggesting that the brain is under immunological surveillance. It has been documented that monocytes, and lymphocytes (T and B) both cross the BBB [Andersson et al, 1992, Neuroscience 48: 169±186; Nottet et al, 1996, J Immunol 156: 1284±1295; Hickey et al, 1991, J Neurosci Res 28: 254±260; Knopf et al, 1998, J Immunol 161: 692±701]. Such studies have necessitated a re-thinking of the role of the BBB in immune cell trafficking into the central nervous system.
An important part of the body's defense of bacterial or viral infections is the ability of monocytes and granulocytes to invade the tissue and begin phagocytosis of the foreign material. Indeed, infiltration of monocytes and neutrophils from the bloodstream into the infected tissue is part of the initial immune response. However, there are notable differences in the penetration of monocytes and neutrophils in the BBB and the peripheral microvasculature. First, trafficking of activated leukocytes across the BBB display differences that are dependent on cell type, with monocytes being more capable than neutrophils in passing through the BBB. This was demonstrated in mice injected with various pro-inflammatory stimuli directly into the hippocampus [Andersson et al, 1991, Neuroscience, 42, 169-186; Andersson et al., 1992, Neuroscience 48, 169-182]. Under normal homeostatic conditions, minimal margination and diapedisis (i.e., movement from blood vessels to tissues) of both neutrophils and monocytes was observed in the BBB. Following hippocampal injection of kianic acid or LPS challenge, a dramatic increase in the margination of neutrophils and monocytes to the brain endothelial cells was seen [Andersson et al, 1991, Neuroscience, 42, 169-186; Andersson et al., 1992, Neuroscience 48, 169-182]. Although there was an increased attachment of both neutrophils and monocytes to the brain endothelial cells, only the monocytes underwent diapedisis and were able to move through the BBB into the brain. Infiltration of neutrophils through the BBB was only observed following breakdown of BBB integrity, and was in contrast to the rapid and substantial accumulation of neutrophils observed in the CSF through the choriod plexus [Andersson et al, 1991, Neuroscience, 42, 169-186; Andersson et al., 1992, Neuroscience 48, 169-182]. The relative inability of neutrophils to move across the BBB, suggests that it is the initial monocyte response that is the important mediator of the immune response in the CNS. The observation that the number of macrophage cells in the brain (following LPS injection) was reduced when peripheral circulating monocytes were depleted, suggests that a significant number of the macrophages in the brain parenchyma were attributable to the infiltration of monocytes across the BBB [Andersson et al., 1992, Neuroscience 48, 169-182].
Transport of the immune cells into the CNS is attributed in part, to the adhesion molecules found in the vascular endothelial cells that form the BBB. Some of these adhesion molecules, like the cadherins, are involved in the formation of intercellular tight junction complexes between the brain microvessel endothelial cells.
It should be noted that leukocyte transendothelial migration across an altered BBB is a prominent feature of many neurodegenerative disorders. Marked changes in BBB function is observed in several neurological disorders associated with immune responses such as multiple sclerosis (MS), meningitis, and HIV-1-associated dementia.
It is well known that cells belonging to the MPS, including monocytes and macrophages, are able to phagocytose particles such as liposomes. Surface charge, size, concentration and composition are all known to influence the cell's ability for phagocytosis. According to some research, negatively charged liposomes associate more effectively with the phagocytic cells [Raz, A., et al., 1981, Cancer Research 41, 487-494; Hsu, M., et al., (1982) Biochem. Biophys. Acta 720, 411-419] and large-sized liposomes (above 500 nm) are also phagocytized more efficiently. The use of PEG linked to the lipids comprising the liposomal membrane prevents uptake by the phagocytic cells such that cationic liposomes without PEG are taken up by the reticuloendothelial system (RES) at a much higher rate than PEGylated cationic liposomes are. The same is true for sterically shielded lipids such as ganglioside-GM1 and phosphatidylinositol. Liposomes containing negatively charged lipids that are not sterically shielded (phosphatidylserine, phosphatidic acid, and phosphatidylglycerol) are phagocytosed more readily. Liposomes containing sterically shielded lipids are cleared even more slowly than neutral liposomes [Gabizon A., Papahadjopoulos D. Biochim. Biophys. Acta, 1103: 94-100, 1992]. On the other hand, inclusion of cholesterol in the liposome enhances uptake by the MPS [Ahsan, F. et al., 2002, Journal of controlled Release, 79, 29-40].
It has been suggested that liposomes may cross a disrupted blood brain barrier using a “particle pick-up” mechanism by circulating macrophages [Rousseau et al., 1999, Experimental brain research; Experimentelle Hirnforschung; Experimentation cerebrale 125 (1999) 255-64]. However, the mechanism is dismissed (by the authors themselves) because of the short delay between liposome injection and brain localization as assessed by scintigraphy. If circulating macrophages did indeed capture the liposomes, this would take time to occur and brain activity would progressively increase, which was not the case in Rousseau's findings. Of note, the formulation and size of liposomes used in this study were not selected to encourage phagocytosis by cells of the MPS which also suggests that the liposomes of this study were unlikely to cross the BBB via a macrophages pick-up mechanism. Also, Rousseau envisaged using liposomes to cross the BBB as a diagnostic tool only and not as a therapeutic aid.
In a research carried out by Huitinga et al., [J Exp Med 172 (1990) 1025-33], it is suggested that mannosylated liposomes may cross the blood brain barrier via activated monocytes. He suggests that mannosyl receptors can be expressed on activated monocytes leading to a more efficient in vitro binding of mannosylated liposomes to macrophages. However, this explanation contradicts his findings since, whereas both mannosylated and non-mannosylated liposomes were incorporated in monocytes in the spleen and liver equally well, only the modified liposomes could penetrate the BBB.
In both the studies carried out by Roussea et al, 1999 supra and Huitinga et al, 1999 supra the liposomes were confined to the BBB area and did not move out to other brain areas.
U.S. Pat. Application No. 20050048002 to Barrett discusses the use of phagocytic cells as carriers of particles to the brain, but does not discuss using phagocytic cells as transporters of particles to other organs protected by a barrier including the eye, testicles and mammary gland. Barrett et al., does not relate at all to the use of liposomes as carriers, but rather to solid particles (e.g. a microparticle or nanoparticle) between 150 nm to 100 μm. Barrett et al., does not relate to methods of enhancing uptake by phagocytic cells such as by using negatively charged particles. Indeed, Barrett specifies pegylated phospholipids and sterically shielded lipids (as a surfactant only) both of which are known to prevent uptake by phagocytic cells.
U.S. Pat. Application No. 20040266734 and U.S. Pat. Application No. 20040265391 both describe liposomes of a similar formulation to that of the present invention containing an agent which is toxic to MPS for the treatment of restenosis and acute coronary syndromes, thereby teaching away from the present invention.
In summary, the mechanism of “liposome pick up” by circulating macrophages has been discussed with reference to either a disrupted blood brain barrier or mannosylated liposomes. In both of these the mechanism was deemed unlikely. When discussed in U.S. Pat. Application No. 20050048002, liposomes are not mentioned and the only reference to lipid substances relates to surfactants which may prevent effective uptake by phagocytic cells if pegylated or shielded as described therein.
In view of the above, there is a widely recognized need for and it would be highly advantageous to have an improved method of delivering therapeutic and diagnostic agents across blood barriers and into target tissues.